The Impact of Dermal Characteristics on Low-Level Laser Power Measurement in Postmortem Zoological Species

Photobiomodulation therapy, also termed as low-level laser therapy, is commonly used as an adjunctive therapy for various medical conditions in veterinary practice. The ACTIVet PRO low-level laser has been used for treatment of various nondomestic species, yet the effects of dermal attributes such as pigment, feathers, or scales have not been evaluated. The effects of low-level laser therapy with the ACTIVet PRO have been investigated in laboratory animals, including a study in rats that evaluated the passage of laser light through the skin in postmortem samples. The objective of this study was to measure the power of a low-level laser (ACTIVet PRO) after penetration through dermal tissue (∼1 mm thickness) in a variety of postmortem animal tissue. This study sought to determine the impact of fur, feathers, scales, and different pigments on the ability of the laser to penetrate. Frozen and thawed skin tissue samples from various species were placed inside a light restricted laser box and exposed to a preprogrammed laser level from a Multi Radiance ACTIVet PRO photobiomodulation (PBM) device, with a power meter to measure the light penetration through the tissue samples. Light penetration measurements via power output measurements (mW) were recorded at 7 time points (range, 1–150 sec). A Friedman test was performed to evaluate the difference of the mean tissue penetration by each species at each time point. Lighter colored specimens had higher power readings than darker colored or pigmented samples, and feathers appeared to inhibit the laser, showing minimal to no power readings on bird skin covered in covert and down feathers. There was statistically significant mean tissue penetration for all time points between the rabbit and green sea turtle (p=0.0046), the red-tailed hawk and green iguana (p=0.0046), and the red-tailed hawk and green sea turtle (p=0.000034). Overall findings found that certain skin coverings, such as feathers, appear to inhibit passage of laser light through tissue to the photo meter. Darker pigmented areas of tissue appeared to absorb the laser light, which also did not allow light passage through the tissue to the photo meter. All of this illustrates that there are differences in tissue penetration between different animal species, at least in postmortem tissues. This could necessitate adjustment of machine settings for therapeutic effect in different species, though further studies would be warranted to determine the extent to which this would be necessary. Additional studies evaluating biologically active tissues would be needed as a next step, as photobiomodulation has an effect at the cellular level and the exact amount of medical benefit is not measurable in skin samples that are separate from a living organism.


Introduction
Te acronym LASER stands for "light amplifcation by stimulated emission of radiation" [1], which is now commonly used as a noun.In veterinary medicine, lasers are commonly used for a variety of surgical and therapeutic procedures.Photobiomodulation therapy (PBMT) is a common form of laser use in the veterinary feld, which "results in benefcial therapeutic outcomes including but limited to the alleviation of pain or infammation, immunomodulation, and promotion of wound healing and tissue regeneration" [2][3][4][5].
Te ability of a laser to penetrate tissue depends on the wavelength of the laser [6].Wavelengths that minimize light scattering and refection at the surface of the tissues and absorption by chromatophores are imperative for optimal tissue penetration [7].Te targeting of deeper cells for therapy requires lasers to emit wavelengths in the 800-1000 nm range, which is considered the "therapeutic" or "optical" window for PBMT as this range allows for chromophores within tissue to absorb light [8].Laser light also emits three wavelengths that allow it to penetrate through tissues and down to the cellular level [8].Tese include monochromatic (single-color wavelength), collimated (nondivergent) and coherent (in-phase wavelengths) light [9].Te monochromatic wavelength allows for photon emissions from one set anatomic energy level.Te collimated wavelength achieves a narrow beam, and the coherent wavelength allows for photons traveling in phase with each other [8].
Tere have not been many studies performed that would determine what efect fur, feathers, scales, and skin pigment have on the penetration of PBMT light into tissue [6].Additionally, there are very few studies demonstrating the use of PBMT in these species [10,11].We believe that there are factors that could infuence the efcacy of PBMT such as tissue thickness/density, location on the body, the therapy is being performed on, tissue type, tissue pigment and hemoglobin concentration, antemortem vs. postmortem tissue, and temperature.We hypothesized that the power measurement would be greatest in tissues that lack pigment (e.g., white fur and pale skin) and/or that lack a material that could disperse or impede the passage of the PBMT light wavelength (e.g., feathers or thick scales).

Animal Samples.
Postmortem samples of 10 rabbits (Oryctolagus cuniculus), 12 guinea pigs (Cavia porcellus), 6 green iguanas (Iguana iguana), 11 American bullfrogs (Lithobates catesbeianus), 13 red-tailed hawks (Buteo jamaicensis), 2 green sea turtles (Chelonia mydas), 22 Eastern Box turtles (Terrapene carolina carolina), and 1 Kemp's Ridley sea turtle (Lepidochelys kempii) were used for this project (sea turtle samples collected under NC Wildlife Commision Endangered Species permit 18ST42).All samples were frozen and then thawed under refrigeration (40 degrees Fahrenheit) prior to use for this project.Dermal dissection was performed using a #11 scalpel blade with a linear incision medially, preserving the dorsal and anterior surfaces of the stife region, and extending from the mid-femur to mid-to-distal tibia, yielding 4-6 cm of irregularly shaped (though most closely resembling rectangles).Samples were used immediately after sectioning of the postmortem specimen.Each skin sample was exposed to the laser only once due to biological transformation of the tissue.Skin color, skin thickness (measured with digital callipers), and temperature (determined by infrared thermometer (10 : 1 Infrared Temp Gun, Milwaukee Tool, Brookfeld, WI 53005) on the margin of the tissue to avoid the location of the PBMT) were recorded for each sample.Samples were analyzed in the same room under standard room temperature of 70 degrees Fahrenheit.
Of the 10 rabbits, two samples were taken from 9 animals, one sample from each leg.12 guinea pigs were used with two samples taken from each animal.For the red-tailed hawk specimens, two samples were taken from 8 of the 13 specimens.Two samples were taken from the one Kemp's Ridley sea turtle and both green sea turtles.Two samples were taken from the 11 American bullfrogs.Only one of the Eastern Box turtles had two samples.One sample from six green iguana specimens was used.

Instruments. An MR4 ACTIVet PRO (Multi Radiance
Medical, Solon, OH 44139) low-level laser 1000 Hz (program 3, 905 nm) laser was used for this study.Te laser was set to on for 5 minutes at 250 Hz prior to any sample collection.
Te Optical Power Meter System (Torlabs Instruments, Newton, NJ) was used to measure laser mean power output.Te optical power meter system consisted of a PM200 console display unit with a 6 Hz sample rate and ± 1% accuracy.Te S322C power meter sensor had a 4 cm 2 aperture area with an optimal power range from 100 uW to 1.0 W. Power meter calibration was performed just prior to the frst sample to ±1% and minimal variation was expected over the time period.
A stand for the laser was constructed to maintain constant distance of the laser to the power meter (Figure 1).Te power meter was secured in place with three pieces of transparent packing tape, which was housed within a circular cut-out within a Styrofoam platform that allowed exposure of the fat surface of the sensor (Figure 1(a)).Te entire Styrofoam surface was then covered in saran wrap.Four external walls comprised a cardboard box surrounded the Styrofoam platform with a square cut out for the handle of the laser.Great Stuf Gaps and Cracks Insulating Spray Foam Sealant was used to specifcally construct a moulded stationary rest for placement of the laser to achieve repeatable 90-degree alignment and direct contact of the convex lens of the laser head with the dermal tissue sample (Figure 1(b)).A thick black garbage bag was placed over the entire set up to eliminate possible infuence of ambient fuorescent lighting (Figure 1(c)).

Statistics.
To evaluate the diference of the mean tissue penetration by each species at each time point, the Friedman test, a nonparametric analysis of variance (ANOVA), was performed.

Data Collection.
Prior to each measurement, the power meter was zeroed after the tissue sample was in place prior to applying the laser.Approved laser safety glasses were worn at all times while the laser was being operated.All measurements were taken in an enclosed climate-controlled room (temperature 68-72 °F).

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Results
Te main fndings of this experiment were that the color of the tissue integument appears to impact power readings as illustrated in Figure 2 for mean tissue penetration by species, as well as mean tissue thickness by species as noted in Table 1.In rabbits, their white nonpigmented skin had the highest power readings, with an average of 14.98 mW, while the grey and fawn rabbits had much lower readings (on average 0.92 mW and 0.75 mW, respectively).Te black rabbit skin had almost no penetration measured (average of −0.05 mW) (Table 2).Similarly, in iguanas and bullfrogs, the lighter colored/pigmented specimens had higher power readings than the darker pigmented integument specimens, with an average reading of 22.1 mW in lighter iguana samples and 33.35 mW in lighter bullfrog samples.Conversely, the darkest iguana integument samples had an average reading of 16.42 mW and the darkest bullfrog integument samples averaged 16.32 mW.In sea turtles, Kemp's Ridley skin had higher power readings than the green sea turtle skin (average 36.88 mW and 23.16 mW, respectively).
Statistically, there was a signifcant diference in mean tissue penetration for all time points between the rabbit and green sea turtle (p � 0.0046), the red-tailed hawk and green iguana (p � 0.0046), and the red-tailed hawk and green sea turtle (p � 0.000034).

Discussion
Te therapeutic outcome of PBM is the result of inducing various cellular changes, with systemic efects that include decreased infammation via stimulation of immune cells with specifc wavelengths of light, which releases infammatory mediators [8,12].Tese are important in wound healing, pain control, and nervous system injury, among others [8].Previous studies have not fully determined the efect fur, feathers, scales, and skin pigment have on the penetration of low-level laser light into tissues despite evaluating for clinical relevance [6,10,11], and thus, it is unknown what degree of tissue penetration occurred in these studies.In these studies, it was believed that a clinical diference or a factor of healing was observed, so it is presumed that the PBMT was passing through the tissues and having a biological efect [10,11].Although we were unable to determine biological efect directly in this study of postmortem tissues, we can compare to these publications and that with tissue penetration biological activity should be able to be observed, although the extent of biological activity and power needed would require additional research.Te bioluminescent green sheen on the inside of the skin of green sea turtles may have impacted the ability of the laser light to penetrate to the optical power meter.Te same pigment trend was observed in box turtles, and although there was also a green sheen noted on some of these samples, they had a much lower average tissue penetration regardless of sample pigment/color.In red-tailed hawks, there was minimal to no power readings, other than when the feathers were compressed, in which case the power readings were higher.Tis observation appeared to be an actual fnding, but additional studies could be performed to further clarify this.Te fndings in our study support our hypothesis that Veterinary Medicine International pigmented and darker tissues would have lower power readings.Tis could indicate that darker pigmented tissues do not allow the laser light to pass as readily or as deeply compared to more lightly pigmented tissues.Additionally, other attributes such as feathers and scales did negatively impact the power output readings, as predicted.Te signifcant diference in penetration between the: rabbit and green sea turtle, the red-tailed hawk and green iguana, and the red-tailed hawk and green sea turtle further illustrated that there are diferences in tissue penetration between diferent animal species and their postmortem tissue samples.
A potential bias of this study could be the use of postmortem samples.Tissue penetration measurements may be diferent in living versus nonliving tissue, such that circulating blood in living skin tissue may alter the penetration or the absorption of the laser light.Tissues were thawed prior to the study, so tissue would be less likely to cause refraction of the laser when compared to frozen samples, though it could also be possible that retained water in the tissues could have afected this study.Because the cells in these samples were dead and an enhancement of the PBM mechanisms was not possible, the actual biological activity or medical beneft of the PBMT unit could not be evaluated as a part of this study.Tere is increased variability of the laser itself when used over long time periods, which could produce overheating and thermal drift, although care was taken to not use the laser for long time periods during data collection sessions.Body condition of the tissues could be another potential limitation, as there are diferences between muscle, fat, and water content in the tissue samples.A diference was noted in the absorption of animals with extensive down and covert feathers such as the red-tailed hawk.Te feathers on one hawk sample were compressed, which allowed the skin tissue to have more direct exposure to the laser light and allowed for much greater power readings compared to the noncompressed down and covert feathers.Additionally, for the samples where there was no power reading due to the darker pigment of the animal's integument or the sheen of the tissue, it is possible that instead the light was being absorbed and not passing through the integument.Another potential could be that the light was refracted, although we were unable to determine which scenario was occurring.As we were using postmortem tissues, however, we are not able to determine if this would lead to a higher PBMT efect or not.
Although some tissues did not appear to have tissue penetration measurements recorded in this study, it is still possible that they could have a metabolic beneft and that the use of PBMT should not be discouraged.Additionally, due to the diferences in penetration, ideally, if the animal has feathers, a greater penetration may be achieved if the feathers are compressed or moved out of the way prior to PBMT use.
Overall, the clinical relevance of diferences in tissue penetration in zoological species found in this study is difcult to determine based on using postmortem tissues.We would recommend further assessment to determine if the lower or higher level of penetration relates to therapeutic outcomes.Additional research could be done to determine if darker pigmented tissues have a greater overall positive therapeutic efect at the same setting as a lighter pigmented tissue.Further studies are also recommended in determining clinical relevance of live tissue and assess penetration levels.

Conclusions
Photobiomodulation therapy is becoming a common practice in veterinary medicine.Tis study was designed to evaluate the tissue penetration for PBMT light and found that lighter-pigmented tissues appear to allow for higher power output readings and thus increased laser light penetration compared to darker pigmented tissue.Also, it appears that noncompressed feathers may hinder the laser light penetration and have lower tissue penetration levels.Given the limitation of using postmortem tissues in this study, results should be assessed taking this into consideration, and further research is necessary to determine the signifcance of diferences in laser penetration between species and skin coverings.Regardless of the readings we have reported, PBMT has been reported to have systemic efects and metabolic benefts overall; therefore, low-level laser therapy may still be benefcial, even with little or no power readings.

Figure 1 :
Figure 1: A hand-constructed stand was used to facilitate power measurements of the ACTIVet PRO photobiomodulation laser through postmortem dermal tissue of multiple zoological species.(a) A Styrofoam platform with circular cut-out illustrating a rabbit skin sample overlaying the optical power meter.(b) A cardboard box surrounding fxed spray foam holding an ACTIVet PRO photobiomodulation laser in place over a tissue sample and optical power meter.(c) Te complete stand set-up with optical power meter connected to computer for data collection and covered with an opaque garbage bag to eliminate ambient fuorescent light.

Figure 2 :
Figure 2: Mean tissue penetration measured via mean power output (mW) of an MR4 ACTIVet PRO photobiomodulation laser (905 nm) at predetermined exposure time points by species (American bullfrog, eastern box turtle, green iguana, green sea turtle, domestic guinea pig, Kemp's Ridley sea turtle, domestic rabbit, and red-tailed hawk) with standard error bars at each exposure time point.

Table 1 :
Mean tissue thickness (mm) by species of dermal samples that were analyzed by an MR4 ACTIVet PRO photobiomodulation laser.